The accurate replication of duplex DNA requires the coordinated activity of many types of proteins. We are studying these enzymatic reactions and protein-protein interactions using a model system of highly purified bacteriophage T4 encoded proteins. Efficient DNA replication in vitro is achieved with T4 DNA polymerase (gene 43), gene 32 DNA helix-destabilizing protein, the gene 44/62 clamp loader, gene 45 polymerase clamp, gene 41 helicase, gene 59 helicase loading protein, gene 61, primase, RNase H, DNA ligase, and DNA topoisomerase. 41 Helicase can load on DNA by itself, but its loading is accelerated by T4 59 protein, particularly on DNA that is covered with T4 32 ssDNA binding protein. During the past year we have continued our studies of the mechanism by which the 59 helicase-loading protein loads the helicase. Phage T4 uses two modes of replication initiation, origin-dependent replication from several different origins early in infection and recombination-dependent replication at later times. The same relatively simple complex of T4 replication proteins is responsible for both modes of DNA synthesis. Thus the mechanism for loading the T4 41 helicase must be versatile enough to allow it to load on R loops created by transcription at origins with different sequences, on D loops created by recombination, and on stalled replication forks. We have previously shown that 59 protein binds to fork DNA, four-stranded Holliday junctions, and three-stranded invasion structures, and stimulates 41 helicase activity on each of these DNA structures. We are currently investigating how this helicase activity differs on mobile and nonmobile Holliday junctions. The crystal structure of 59 helicase loading protein, solved by our collaborators Timothy Mueser and Craig Hyde, revealed a novel, almost entirely -helical protein, with no similarity to the structures of other single-stranded DNA binding proteins. The N-terminal domain of 59 protein has strong structural similarity to several members of the high-mobility-group (HMG) family proteins. We proposed a speculative model for 59 protein on a replication fork DNA, based on the assumption that the "HMG-like" N-domain binds the duplex ahead of the fork and holds the beginning of the fork arms in an open configuration to provide docking areas for 41 helicase and 32 single-stranded DNA binding protein. In support of this model, we find that the I87A mutation in 59 protein reduces fork binding and helicase stimulation, while deletion of the C-terminal two residues KY decreases the ability of 59 protein to promote binding of 32 protein on small fork DNA. Two bacteriophage T4 origins of replication, (ori(uvsY) and ori(34)), have been shown by Ken Kreuzer and coworkers to consist of a T4 middle-mode promoter juxtaposed to a DNA unwinding element (DUE). In collaboration with Kreuzer (Duke University) we have established a system of T4 replication proteins that catalyze complete unidirectional replication of a plasmid containing the T4 ori(uvsY) origin, with a preformed R loop at the position of the R loop identified at this origin in vivo. This replication is dependent on the 41 helicase and strongly stimulated by 59 protein. Moreover, the helicase loading protein helps to coordinate leading and lagging strand synthesis by blocking replication on the ori(uvsY) R loop plasmid until the helicase is loaded. The T4 enzymes can also replicate plasmids with R loops that do not have a T4 origin sequence, but only if the R loops are within an easily unwound DNA sequence. Thus, the requirement for loading the T4 replication enzymes, including the helicase and 59 helicase loading protein, is not a specific T4 sequence, but rather a specific structure, an R loop or D loop within an easily unwound DNA sequence. T4 RNaseH is a 5' to 3' exonuclease that is a member of the RAD2 family of eukaryotic and prokaryotic replication and repair nucleases. We have previously shown that this nuclease removes the pentanucleotide RNA primers and 10-50 nucleotides of adjacent DNA from each discontinuous lagging strand fragment on the DNA replication fork in vitro. We are continuing our studies to determine how the activity of this nuclease is modulated by its interactions with 32 protein and the gene 45 clamp protein. The gene 45 clamp has been shown by others to interact with T4 DNA polymerase, and with the T4 gene 33 and 55 late transcription proteins through a similar sequence at the C-terminus of each of these proteins. In contrast, we have identified hydrophobic residues at the N-terminus of the nuclease that are important for its stimulation by the polymerase clamp. However, the N-terminal sequence of the nuclease has similarity to the sequence at the C-terminus of the other proteins, suggesting that they may all contact the same region of the clamp. We are collaborating with Jack Griffith (University of North Carolina) to characterize the path of DNA and the structure of proteins on T4 replication forks by electron microscopy. Helicase, unwinding the duplex ahead of the fork, will lengthen the region of single-stranded DNA behind the last discontinuous (Okazaki) fragment on the lagging strand. There will be a second region of ssDNA ahead of this fragment, if the fragment is still being elongated by polymerase. Our studies suggest that the ssDNA on the lagging strand is in a compact, protein-covered form. One or two regions of single-stranded DNA are visible, as expected, on deproteinized replication forks. However, micrographs of forks with the replication proteins show large protein complexes and a double-stranded DNA loop, but no extended ssDNA. We have begun using visible pointers that recognize biotin-labeled T4 proteins to determine the location of proteins in the complex on the replication fork, and to identify the proteins bound to ssDNA in the compacted structures.